WO2008023671A1 - Procédé d'amplification poussée d'un gène cible dans une cellule de mammifère et vecteur correspondant - Google Patents

Procédé d'amplification poussée d'un gène cible dans une cellule de mammifère et vecteur correspondant Download PDF

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WO2008023671A1
WO2008023671A1 PCT/JP2007/066133 JP2007066133W WO2008023671A1 WO 2008023671 A1 WO2008023671 A1 WO 2008023671A1 JP 2007066133 W JP2007066133 W JP 2007066133W WO 2008023671 A1 WO2008023671 A1 WO 2008023671A1
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seq
polynucleotide
base sequence
gene
locus
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PCT/JP2007/066133
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English (en)
Japanese (ja)
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Noriaki Shimizu
Toshihiko Hashizume
Masashi Shimizu
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Hiroshima University
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Priority to KR1020097004529A priority Critical patent/KR101093835B1/ko
Priority to JP2008530901A priority patent/JP5124737B2/ja
Priority to US12/438,545 priority patent/US8137963B2/en
Priority to EP07792746A priority patent/EP2058391B1/fr
Priority to CA2661647A priority patent/CA2661647C/fr
Priority to DE602007009549T priority patent/DE602007009549D1/de
Publication of WO2008023671A1 publication Critical patent/WO2008023671A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/80—Vectors comprising a special origin of replication system from vertebrates
    • C12N2820/85—Vectors comprising a special origin of replication system from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island

Definitions

  • the present invention relates to a method for highly amplifying a target gene in mammalian cells, and a vector used for carrying out the method. More specifically, when a desired gene is amplified using the “advanced gene amplification system” developed by the present inventors, a method capable of improving gene transfer efficiency and gene amplification efficiency, and the method It relates to the vector for performing.
  • the present inventor has derived a plasmid having a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) (hereinafter referred to as "IR / MAR plasmid") from a human. Introduce into cancer cells (COLO 320 colorectal cancer cell line and HeLa cell line) by lipofusion method and select using drug resistance gene (Blasticidine or Neomycine) present on plasmid just
  • target gene The ability to amplify the number of intracellular copies of a gene encoding a protein to be expressed (hereinafter referred to as “target gene” where appropriate) to about 10,000 copies;
  • the target gene is highly amplified regardless of whether it is introduced as the same gene construct (cis) or introduced as a separate gene construct (trans) into the IR / MAR plasmid. It was discovered that it is possible (see Patent Document 1 and Non-Patent Document 1). Then, based on this finding, the present inventor used an IR / MAR plasmid and a target gene for mammalian cells (for example, human-derived cancer cells (COLO 320 colorectal cancer cell line and HeLa cell line), CHO cells, System that can amplify the target gene to about 10,000 copies simply by using the drug resistance gene (Blasticidine or Neomycine) present on the plasmid. ”)" was completed. [0003] Here, Fig.
  • Step 1 shows the mechanism generated by DM and HSR force SIR / MAR plasmid (also called "IR / MAR vector”! / ⁇ ).
  • the IR / MAR plasmid multimerizes by repeatedly connecting in series in the host cell (Stepl). This multimer is stably present in the host cell during cell growth and self-replicates.
  • DM is generated when the multimer grows as it is or is taken up by the DM that originally exists in the host cell.
  • Step 2 multimeric circular DNA is broken into double strand breakage (DSB) in the host cell to become linear DNA. Then, the linear DNA is incorporated into the chromosome, a BFB (Breakage-Fusion-Bridge) cycle like Step 3 is started, and HSR is generated.
  • DSB double strand breakage
  • Patent Document 1 Japanese Published Patent Publication “Japanese Unexamined Patent Publication No. 2003-245083 (Released date: September 2, 2003)”
  • Patent text l3 ⁇ 4 l Noriaki Shimizu, et al. (2001) Plasmids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplificat ion. Cancer Research vol.o ⁇ , no.19, p6987_6990.
  • MAR is a polynucleotide of several hundred bp.
  • the force IR is several kbp.
  • IR from the c-myc locus is 2.4 kbp
  • IR from the dihydrofolate reductase (hereinafter referred to as “DHFR”) locus is 4.6 kbp. Therefore, the IR / MAR plasmid is a relatively large size gene construct.
  • the present invention aims to further improve the advanced gene amplification system. More specifically
  • the object of the present invention is to develop a vector capable of enjoying the merits (A) to (C) and to provide a method for amplifying a target gene using the vector.
  • the present invention includes the following inventions in order to solve the above problems.
  • the method according to the present invention is a method for amplifying a target gene
  • the mammalian replication initiation region may be any one of the c myc locus, the dihydrofolate reductase locus, and the replication initiation region of the / 3-globin locus. It may be derived from.
  • the method according to the present invention is characterized in that the amplification active fragment is derived from the cmyc locus and includes at least a Duplex Unwinding Element and a topoisomerasell binding region. Also good.
  • the amplification active fragment is derived from the C myc locus, and comprises the following polynucleotide (a) and the following (b):
  • the method may be: (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, or a polynucleotide in which one or several bases have been deleted, substituted or added in the base sequence represented by SEQ ID NO: 1;
  • a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 or a polynucleotide in which one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 2.
  • the amplification active fragment may be one or several in the polynucleotide comprising the nucleotide sequence IJ represented by SEQ ID NO: 3 or the nucleotide sequence represented by SEQ ID NO: 3.
  • the polynucleotide may contain a polynucleotide in which the base is deleted, substituted, or added.
  • the amplification active fragment may be one or several in the polynucleotide consisting of the base sequence IJ shown in SEQ ID NO: 4 or the base sequence shown in SEQ ID NO: 4.
  • the polynucleotide may contain a polynucleotide in which the base is deleted, substituted, or added.
  • the amplification active fragment may be one or several in the polynucleotide comprising the nucleotide sequence IJ represented by SEQ ID NO: 5 or the nucleotide sequence represented by SEQ ID NO: 5.
  • the polynucleotide may contain a polynucleotide in which the base is deleted, substituted, or added.
  • the amplification active fragment is derived from the dihydrofolate reductase gene locus and is a polynucleotide comprising the base sequence shown in SEQ ID NO: 10, or the base sequence shown in SEQ ID NO: 10.
  • a polynucleotide comprising one or several bases deleted, substituted, or added may be used.
  • the amplification active fragment is derived from the dihydrofolate reductase gene locus, and is a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 11 or shown in SEQ ID NO: 11 It may be a method characterized by comprising a polynucleotide in which one or several bases are deleted, substituted, or added in the base sequence.
  • the mammalian nuclear matrix-binding region comprises a nuclear matrix-binding of Ig ⁇ locus, SV40 initial region, and dihydrofolate reductase locus. It may be derived from any force in the region.
  • the method according to the present invention may be a method characterized in that the target gene and the vector are arranged in trans and introduced into a mammalian cell.
  • the vector which is effective in the present invention is a vector for amplifying a target gene in a mammalian cell, and is a partial fragment of a mammalian replication initiation region and has a gene amplification active site. It comprises a vector comprising an amplified active fragment, a mammalian nuclear matrix binding region, and a gene for selecting transformed cells.
  • the vector according to the present invention is characterized in that the mammalian replication initiation region is a replication force of the myc locus, the dihydrofolate reductase locus, and the / 3-globin locus! Even if it comes from one! /
  • the vector according to the present invention may be characterized in that the amplification active fragment is derived from the cmyc locus and contains at least a Duplex Unwinding Element and a topoisomerasell binding region.
  • the vector according to the present invention is a vector in which the amplification active fragment is derived from the cmyc locus and includes the following polynucleotide (a) and the following (b): Also good:
  • a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 or a polynucleotide in which one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 2.
  • the amplification active fragment is a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3, or 1 or several nucleotides in the nucleotide sequence represented by SEQ ID NO: 3. It may include a polynucleotide having a base deleted, substituted, or added.
  • the amplification active fragment is 1 in the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4 or the nucleotide sequence represented by SEQ ID NO: 4.
  • it may contain a polynucleotide in which several bases have been deleted, substituted, or added.
  • the amplification active fragment is a polynucleotide having the base sequence represented by SEQ ID NO: 5, or 1 or several nucleotides in the base sequence represented by SEQ ID NO: 5. It may include a polynucleotide having a base deleted, substituted, or added.
  • the amplification active fragment is derived from the dihydrofolate reductase gene locus, and is a polynucleotide comprising the base sequence shown in SEQ ID NO: 10, or shown in SEQ ID NO: 10.
  • the nucleotide sequence may include a polynucleotide in which one or several bases are deleted, substituted, or added.
  • the amplification active fragment is derived from the dihydrofolate reductase gene locus and is a polynucleotide comprising the base sequence represented by SEQ ID NO: 11, or represented by SEQ ID NO: 11.
  • the nucleotide sequence may include a polynucleotide in which one or several bases are deleted, substituted, or added.
  • the vector according to the present invention includes any one of the above-mentioned mammalian nuclear matrix-binding region force S, Ig ⁇ gene locus, SV40 initial region, and nuclear matrix-binding region of the dihydrofolate reductase locus. It may be derived from one.
  • the amplification active fragment is not limited to the polynucleotide specified by the nucleotide sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, 10, and 11. It may be composed of a base sequence complementary to the base sequences shown in 1, 2, 3, 4, 5, 10, and 11.
  • the amplification active fragment is a polynucleotide identified by the nucleotide sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, 10, and 11, and SEQ ID NOs: 1, 2, 3, 4, 5,
  • polynucleotides that hybridize under stringent conditions with a! / A shift force of a polynucleotide having a nucleotide sequence complementary to the nucleotide sequences shown in 10, and 11.
  • stringent conditions means that a hybrida is present only when at least 90% identity, preferably at least 95% identity, most preferably 97% identity exists between sequences. This means that an occurrence will occur.
  • the transformant according to the present invention is effective for the above-described present invention, and a target.
  • a gene is introduced into a mammalian cell.
  • FIG. 1 is a schematic diagram showing the mechanism by which DM and HSR are generated by an IR / MAR plasmid.
  • FIG. 2 Fluorescence microscopic images of DM and HSR detected by FISH method in COLO 320DM transformed with vector, A and B show the results of transformed cells into which pSFVdhfr has been introduced, and C is ⁇ ⁇ ⁇ 2. Shows the result of the transformed cell into which dhfr was introduced, D shows the result of the transformed cell into which pTH2.dhfr was introduced, and E shows the result of the transformed cell into which pEP II was introduced.
  • FIG. 3 is a schematic diagram of a plasmid used in Examples and Reference Examples.
  • 6 is a bar graph showing the frequency of HSR occurring in DM.
  • ⁇ —1, or ⁇ 3 6 is a bar graph showing the frequency of HSR occurrence of HeLa (FIG. 5 (A)), COLO 320HSR (FIG. 5 (B)), or COLO320DM (FIG. 5 (C)).
  • FIG. 6 shows the results of Example 1,
  • A is a schematic diagram of the c myc locus (GenBank HSMYCC; acession number X00364), and B and C are the IR full length of the c myc locus and parts thereof. It is a figure which shows the positional relationship with the fragment
  • FIG. 7 is a diagram showing the results of Example 2, wherein A is a schematic diagram of the DHFR locus Ori- ⁇ region (Genbank CFORIDHFR; accession number X94372), and B is the full-length IR of the DHFR locus Ori- ⁇ region and its FIG. 4 is a diagram showing the positional relationship with partial fragments C0 to C11, and a diagram showing the frequency of HSR occurrence in transformed cells into which a plasmid containing each partial fragment has been introduced.
  • A is a schematic diagram of the DHFR locus Ori- ⁇ region (Genbank CFORIDHFR; accession number X94372)
  • B is the full-length IR of the DHFR locus Ori- ⁇ region
  • FIG. 4 is a diagram showing the positional relationship with partial fragments C0 to C11, and a diagram showing the frequency of HSR occurrence in transformed cells into which a plasmid containing each partial fragment has been introduced.
  • FIG. 8 A is a schematic diagram of the pC12 vector used in the Examples, and B is in the Examples.
  • FIG. 3 is a schematic diagram of the pC12.Psv40 vector used in the experiment.
  • FIG. 9 is a bar graph showing the amount of antibody produced in each experimental group. “With No IR / MARJ shows the results when the IR / MAR plasmid was not transfected,“ ⁇ ⁇ B N.AR1 "Shows the result when cotransformation of p A BN.ARl, and” pC12.Psv 40 “shows the result when cotransformation of pC12.Psv40.
  • FIG. 10 is a bar graph showing the antibody production amount of each clone when pC12.Psv40 is co-transformed in Example 3.
  • FIG. 11 is a bar graph showing the amount of antibody produced by each clone when pABN.AR1 is co-transformed in Example 3.
  • One embodiment of the present invention relates to a method for amplifying a gene of interest.
  • the above method is referred to as “the gene amplification method of the present invention”.
  • the gene amplification method of the present invention comprises:
  • introduction process a method including “introduction process”.
  • target gene means a gene encoding a protein to be expressed.
  • the target gene is not particularly limited, and a polynucleotide encoding a desired protein may be appropriately selected and employed.
  • the target polynucleotide can be obtained using a known technique such as PCR based on the nucleotide sequence information.
  • the gene of interest Amplification is performed on the lumniute chromosome (hereinafter referred to as “DM” as appropriate) and / or in a homogeneously stained region of the chromosome (hereinafter referred to as “HSR” as appropriate).
  • DM lumniute chromosome
  • HSR homogeneously stained region of the chromosome
  • the well-known FISH method fluorescence in
  • Judgment can be made by performing in situ hybridization and detecting the gene introduced into the mammalian cell.
  • a specific method for carrying out the FISH method is not particularly limited, and a conventionally known method may be appropriately selected and adopted.
  • the effect of improving the amplification efficiency of the target gene can be obtained as compared with the conventional advanced gene amplification system, which is more than expected by those skilled in the art (Examples). checking). Whether the amplification efficiency of the target gene has increased or not is determined by the gene amplification structure occurrence frequency (for example, HSR occurrence frequency, DM occurrence frequency) when the conventional advanced gene amplification system is implemented, and the gene amplification method of the present invention. Can be determined by comparing the frequency of gene amplification with the occurrence of the structure.
  • the gene amplification structure occurrence frequency for example, HSR occurrence frequency, DM occurrence frequency
  • the frequency of gene amplification structure generation in the latter is higher than that in the former, the latter, that is, the gene amplification method of the present invention, improves the amplification efficiency of the target gene as compared with the conventional advanced gene amplification system. Then you can confirm!
  • the method according to the present invention includes
  • selection step A step of separating mammalian cells into which the target gene and vector have been introduced (hereinafter referred to as “selection step”) and a culture step of culturing the mammalian cells selected by the selection step (ie, transformed cells) ( Hereinafter, it may be referred to as “culture step”.
  • culture step a culture step of culturing the mammalian cells selected by the selection step (ie, transformed cells)
  • culture step a method for purifying the target protein produced by the above culture step
  • purification step a method for purifying the target protein produced by the above culture step
  • an amplification active fragment which is a partial fragment of a mammalian replication initiation region and has a gene amplification active site, a vector comprising a mammalian nuclear matrix binding region, and a gene for selecting a transformed cell; and the target gene In a mammalian cell.
  • the mammalian replication initiation region and the mammalian nuclear matrix-binding region contained in the vector are a replication initiation region that functions in eukaryotic cells including mammals. There is no particular limitation as long as it is a nuclear matrix binding region.
  • Examples of the mammalian replication initiation region include replication initiation regions derived from the c myc locus, the dihydrofolate reductase (DHFR) locus, the / 3_globin locus, and the like.
  • the replication initiation region derived from the myc locus refers to “McWhinney, C. et al., Nucleic Acids Res. Vol. 18, pl 233_1242 (1990
  • For the replication initiation region of the dihydrofolate reductase locus see “Dijkwel, PA et al., Mol. Cell. Biol. Vol.8, p5398-5409 (1988)”. See also “Aladjem, M. et al., Science vol. 281, pl005-1009 (1998)” for the replication initiation region of the / 3 -globin locus.
  • Examples of the mammalian nuclear matrix-binding region include polynucleotides derived from nuclear matrix-binding regions such as the Ig ⁇ locus, the SV40 initial region, and the dihydrofolate reductase locus.
  • nuclear matrix-binding region of the Ig ⁇ locus see “Tsutsui,. Et al., J. Biol. Chem. Vol. 268, pl 2886 — 12894 (1993)”. See also “Pommier, Y. et al., J. Virol., Vol 64, p419-423 (1990)” for the nuclear matrix binding region of the SV40 initial region. See also Shimizu N. et al., Cancer Res. Vol. 61, P6987-6990 for the nuclear matrix binding region of the dihydrofolate reductase gene locus.
  • IR The mammalian replication initiation region
  • MAR mammalian nuclear matrix binding region
  • IR / MAR plasmid not only full-length IR and MAR but also a vector composed of these partial fragments is also referred to as “IR / MAR plasmid”.
  • an amplification active fragment that is a partial fragment of a mammalian replication initiation region (IR) and has a gene amplification active site is used. It is characterized by.
  • partial fragment of mammalian replication origin region means a part of IR excluding full-length IR.
  • the length of the IR partial fragment is not particularly limited, but in the case of IR derived from the c-myc locus, which is about 2.4 kbp, the length of the partial fragment is 0.5 kbp or more and 2. Okbp or less. It is preferably 0.5 kbp or more and 1.5 kbp or less, and more preferably 0.5 kbp or more and 1.3 kbp or less.
  • the length of the partial fragment is preferably 1.7 kbp or more and 3.5 kbp or less. 1.7 kbp or more and 3. lkb p or less More preferably. When the preferable range is satisfied, the effects (A) to (D) are easily obtained.
  • gene amplification active site means an element essential for gene amplification in the advanced gene amplification system. For example, whether or not an IR partial fragment has the above gene amplification active site can be determined by, for example, preparing an IR / MAR plasmid using the partial fragment and MAR, It can be judged by examining the frequency of gene amplification (HSR, DM) when genes are introduced into mammalian cells.
  • HSR gene amplification
  • gene amplification active sites can be identified by preparing an IR delay mutant and conducting the above examination.
  • Delay mutants can be prepared from IR sequence information by PCR or restriction enzyme digestion.
  • the Duplex Unwinding Element (hereinafter referred to as "DUE") and the topoisomerasell binding region are gene amplification activities. It was found to correspond to a sex site. Therefore, the target gene can be highly amplified by using a partial fragment of IR containing at least the DUE and topoisomerasell binding regions instead of full-length IR in the IR / MAR plasmid.
  • the above IR partial fragment may consist only of the DUE and topoisomerasell binding regions derived from the c-myc locus, or may be a combination of a plurality of DUE and topoisomerasell binding regions. It may be a partial fragment containing the DUE and topoisomerasell binding regions of IR derived from the myc locus.
  • Examples of the base sequence of IR derived from the c myc locus include those corresponding to positions 1 to 2349 of Genbank HSMYCC (accession num ber X00364).
  • a preferred embodiment of the IR partial fragment containing DUE is a polynucleotide corresponding to positions 189 to 473 of Genbank HSMYCC (accession number X0 0364).
  • the nucleotide sequence of the above polynucleotide is shown in SEQ ID NO: 1. However, only the force shown in SEQ ID NO: 1 does not correspond to the preferred embodiment of the partial fragment of IR containing DUE.
  • the added polynucleotide ie, the mutated polynucleotide
  • the mutated polynucleotide also corresponds to a preferred embodiment of the IR partial fragment containing DUE. It should be noted that when an IR / MAR plasmid is constructed using the above-mentioned mutant polynucleotide, it has the activity of amplifying the target gene.
  • the base sequence shown in SEQ ID NO: 1 can be subjected to homologous search against databases such as GenBank, EMBL, and DDBJ.
  • the obtained polynucleotide comprising the base sequence can be used as a partial fragment of IR containing DUE.
  • the similarity between the above-mentioned mutant polynucleotide or the polynucleotide obtained by performing homologous search and the polynucleotide shown in SEQ ID NO: 1 is about 80% or more, preferably about 90% or more. More than 95% is most preferred.
  • Genbank HSMYCC accession number X00364
  • Genbank HSMYCC accession number X00364
  • 745 A polynucleotide corresponding to position ⁇ 987.
  • the nucleotide sequence of the above polynucleotide is shown in SEQ ID NO: 2.
  • SEQ ID NO: 2 only those shown in SEQ ID NO: 2 have the ability S, and one or several bases are deleted in the base sequence shown in SEQ ID NO: 2 which does not correspond to the preferred embodiment of the IR partial fragment containing the topoisomerasell binding region Those skilled in the art will readily understand that substituted, added, or mutated polynucleotides also fall within the preferred embodiment of the IR partial fragment containing the topoisomerasell binding region.
  • the mutant polynucleotide is as described above.
  • a preferred embodiment of the IR partial fragment containing at least the DUE and topoisomerasell binding region is a polynucleotide corresponding to positions 189 to 987 of Genbank HSMYCC (accession number X00364).
  • the nucleotide sequence of the above polynucleotide is shown in SEQ ID NO: 3.
  • SEQ ID NO: 3 only the force shown in SEQ ID NO: 3 does not correspond to the preferred embodiment of the partial fragment of IR including at least the DUE and topoisomeras ell binding region, and the nucleotide sequence shown in SEQ ID NO: 3 lacks one or several bases.
  • mutant polynucleotide is as described above.
  • the present inventors have confirmed that the polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 11, 12, 13, 14, 15, and 16 corresponds to the amplification active fragment. . From these results, it is possible to amplify the target gene by using a polynucleotide containing at least the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 instead of the full-length IR of the IR / MAR plasmid. I found.
  • a polynucleotide in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 10, that is, a mutant polynucleotide is also included in the gene amplification method according to the present invention.
  • a mutant polynucleotide is also included in the gene amplification method according to the present invention.
  • DHFR comprising a polynucleotide comprising the base sequence shown in SEQ ID NO: 10, or a polynucleotide in which one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 10.
  • an amplification active fragment derived from SEQ ID NOs: 11, 12, 13, Examples include polynucleotides consisting of the base sequences shown in 14, 15, and 16, and mutant polynucleotides of the polynucleotides.
  • the amplified active fragment contains the DUE and topoisomerasell binding regions, similar to those of the c-myc locus-derived IR.
  • the DHFR-derived IR amplification fragment further contains curved DNA, RIP60 binding region, and AT-rich element.
  • One example is the base sequence shown at positions 1532 to 6166 of X94372).
  • the IR / MAR plasmid used in the introduction step of the gene amplification method according to the present invention is not limited as long as it contains the above-mentioned amplification active fragment and MAR.
  • AR is a self-reliable sequence required for cloning in E. coli, drug resistance gene as a selectable marker (marker one protein) (blasticidin resistance gene, neomycin resistance gene, hygromycin resistance gene, etc.) Alternatively, a green fluorescent protein gene or the like may be included. By using these selectable markers as indicators, mammalian cells introduced with IR / MAR plasmid can be selected.
  • the target gene introduced in the introduction step of the gene amplification method according to the present invention is controllably linked to a promoter.
  • the promoter is not particularly limited as long as it functions in a mammalian cell to be introduced.
  • a promoter (this book) whose transcriptional activity is activated or inactivated by a predetermined operation using a transcription factor or the like.
  • it may be “a transcriptional activity-regulated promoter” or a constitutive promoter in which transcriptional activity is constitutively activated.
  • the “transcriptional activity-regulated promoter” is not particularly limited as long as it has the above-mentioned characteristics.
  • TRE promoter Clontech
  • T-REX promoter Invitrogen
  • the constitutive promoters include CMV promoter, SV40 early region-derived promoter (SV40 promoter), SRalpha promoter (SRa promoter 1) LTR promoter, MMTV promoter, etc. can be used.
  • the IR / MAR plasmid and the target gene are simultaneously introduced into a mammalian cell.
  • the target gene is highly amplified in the legal adduct cell.
  • the mammalian cells are not particularly limited, and examples include CHO-K1 cells (source: ATCC CCL-61, RI EN RCB0285, RI EN RCB0403, etc.), various tumor cells, and the like. .
  • CHO-K1 cells source: ATCC CCL-61, RI EN RCB0285, RI EN RCB0403, etc.
  • various tumor cells and the like.
  • tumor cells having infinite proliferation ability are particularly preferable.
  • tumor cells examples include HeLa cells (obtained from, for example, ATCC CCL-2, ATCC CCL-2.2, RIKEN RCB0007, RIKEN RCB0191, etc.), human colon cancer COLO 320DM cells (obtained from, for example, ATCC CCL- 220), human colorectal cancer COLO 320 HSR cells (source: eg ATCC CCL-220.1), NS0 cells (source: eg RI EN RCB0213), and the like.
  • HeLa cells obtained from, for example, ATCC CCL-2, ATCC CCL-2.2, RIKEN RCB0007, RIKEN RCB0191, etc.
  • human colon cancer COLO 320DM cells obtained from, for example, ATCC CCL- 220
  • human colorectal cancer COLO 320 HSR cells source: eg ATCC CCL-220.1
  • NS0 cells source: eg RI EN RCB0213
  • the IR / MAR plasmid and the gene of interest are not particularly limited as long as they are introduced into mammalian cells at the same time when introduced into mammalian cells.
  • the former is called IR / MAR plasmid and target gene in cis
  • the latter is IR / MAR plasmid and target gene in trans.
  • the operation can be easily performed by introducing one gene construct into a mammalian cell.
  • the advantages (A) to (D) are more easily obtained.
  • the form of the gene construct may be a plasmid or a cosmid.
  • the IR / MAR plasmid and the target gene can be introduced into mammalian cells by appropriately selecting a publicly known method such as lipofection, electopore position method, and particle gun method. .
  • each gene construct contains a gene encoding a selection marker! /.
  • the selection marker contained in the IR / MAR plasmid is preferably different from the selection marker contained in the gene construct containing the target gene.
  • the “selection step” in the gene amplification method of the present invention is a step of separating mammalian cells into which a target gene and a vector have been introduced. More specifically, in this step, a clone of a cell containing a mammalian cell into which the target gene and vector have been introduced! /, Na! /, And a mammalian cell into which the target gene and vector have been introduced is included. This is the process of selecting the latter cells from the sex population.
  • this step may include a step of culturing mammalian cells in a medium.In this step, the target gene and vector are introduced! /, N!
  • the specific method of the selection step is not particularly limited! /, But, for example, a drug resistance gene is used in a gene construct used when introducing a target gene and a vector into a mammalian cell. If so, use the drug resistance to select mammalian cells into which the target gene and vector have been introduced! /.
  • the selection step in the gene introduction method of the present invention can also be performed by detecting a target gene or vector or a nucleotide fragment thereof contained in a mammalian cell by PCR or Southern blotting.
  • Specific methods of the drug resistance, PCR method and Southern plot method are not particularly limited, and known methods can be appropriately used.
  • the “culturing step” in the gene amplification method of the present invention is a step of culturing the mammalian cells already selected by the above selection step.
  • mammalian cells into which the target gene has been introduced and highly amplified can be grown, and the target protein can be produced by a predetermined operation (transcription induction operation or the like).
  • the specific method of the culturing step is not particularly limited, and may be appropriately adopted after examining the optimum conditions for the cultured mammalian cells.
  • the “purification step” in the gene amplification method of the present invention is a method for purifying the target protein produced by the culture step.
  • mammalian cells are suspended in a buffer solution such as PBS (Phosphate Buffered Saline), and then the cells are disrupted with a homogenizer or ultrasonic waves. Centrifuge and collect the supernatant.
  • a surfactant for promoting protein solubilization, a reducing agent for stabilizing the three-dimensional structure of the protein, and a protease inhibitor for preventing protein degradation may be appropriately added. it can.
  • CHAPS ((3-choliaopropyi) _mmethylammomo_l_propanesulfonate Triton X_100 Nikkol n-octylglycoside, etc.
  • DTT dithiothreitol ol DET (dithioerythritol)
  • aprotune or leupeptin can be used as the protease inhibitor.
  • the protein (target protein) encoded by the target gene can be purified using column chromatography such as affinity chromatography, ion exchange chromatography, filtration chromatography, or the like.
  • unnecessary salts can be removed by dialyzing the purified protein solution against an appropriate buffer.
  • the protein purification process described above is preferably performed under low temperature conditions to prevent protein degradation! In particular, it is preferred that the purification process be performed at 4 ° C! /.
  • the present invention also relates to a vector for carrying out the gene amplification method of the present invention described above (hereinafter referred to as “the vector of the present invention”) or a gene amplification kit comprising the vector (hereinafter referred to as “the present invention”). Kit "and! /, U).
  • the description of the IR / MAR plasmid used in the gene amplification method of the present invention can be used.
  • the kit of the present invention is characterized by including the vector of the present invention. As long as the present invention is capable of performing the method according to the present invention, the kit may include other configurations that are not limited to the above configurations. It should be noted that the power S of the present invention will be described with reference to the description of the method according to the present invention as appropriate.
  • the IR gene of the DHFR locus Ori- ⁇ region and the c-myc IR gene having Asc I sites at the 5 ′ and 3 ′ ends are as follows.
  • the DHFR locus Ori— / 3 region IR (4.6 kbp) was restricted with Not I from pSFVdhfr described in “Shimizu, et al. (2001) Cancer Research, vol. 61, p6987_6990”. It was cut out by digestion.
  • the IR (2.4 kbp) of c-myc is from pNeo. Myc—2.4 described in “McWhinney, C. et al., Nucleic Acids Res. Vol. 18, pl233-1242 (1990)”.
  • plasmids described in “ ⁇ ⁇ Shimizu, et al. (2001) Cancer Research, vol. 61, p6987_6990” were used as ⁇ ⁇ AR ⁇ AR1, ⁇ ⁇ ⁇ . PolyA, and pSFV—V, respectively.
  • p ⁇ ⁇ was synthesized by removing the full-length hygromycin resistance gene cassette contained in pSFV-V using Not I and Nru I restriction enzymes, and containing a multicloning site instead. It was made by inserting an oligonucleotide.
  • the restriction enzyme site of the above-mentioned multicloning site is located downstream of the blasticidin resistance gene (hereinafter referred to as “BSR”). From the 5 ′ end to the 3 ′ end, the Kpn I—Not I—Asc I—Nru I In order.
  • ⁇ ⁇ ⁇ ⁇ shown in Fig. 3D was designed so that a gene containing HSV poly A sequence (1357 bp) was added to the 5 'and 3' ends with Kpn I restriction enzyme sites. Amplification was performed by PCR using a primer set of HSVpAKpnIR and HSVpAKpnIL, and inserted into the KpnI site of ⁇ using the KpnI restriction enzyme site.
  • the nucleotide sequences of the HSVpAKpnIR and HSVpAKpnIL primers are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
  • the base sequence of the above HSV poly A sequence is represented by SEQ ID NO: 19.
  • p ⁇ HpAdhfr is the DHFR locus generated above, and the IR of the Ori- ⁇ region is the MAR sequence force BSR transcription start site present in the IR fragment. It was prepared by inserting into the Asc I site of p ⁇ HpA so that it was farther away.
  • ⁇ ⁇ ⁇ 2. dhfr shown in FIG. 3F is a gene fragment containing the above-mentioned HSV poly A arrangement I] (shown as “HSV pA” in FIG. 3) subjected to blunt end treatment. ⁇ HpA. It was made by inserting into the Nru I site of dhfr.
  • pTH.IR.MAR in Fig. 3C was prepared as follows.
  • the MAR gene in the early region of SV40 was obtained by PCR using the SV40L and SV40R primer sets with pNeo.
  • the nucleotide sequences of SV40L and SV40R are shown in SEQ ID NOs: 20 and 21, respectively.
  • the initial area of SV40 The nucleotide sequence of the MAR gene in the region is shown in SEQ ID NO: 22.
  • the gene (1 18 bp) containing the RFB (replication fork barrier) sequence is designed so that NotI restriction enzyme sites are added to the 5 'and 3' ends of pSV2. It was obtained by PCR using a primer set of RFB Not IL and RFB Not IR. Since the RFB sequence blocks the generation of replication forks, it can inhibit the replication of the plasmid into which the RFB sequence has been introduced.
  • the nucleotide sequences of RFB Not IL and RFB Not IR are shown in SEQ ID NOs: 23 and 24, respectively.
  • the base sequence of the RFB sequence is represented by SEQ ID NO: 25.
  • pTH2.dhfr or ⁇ 2 ⁇ dhfr.inv shown in Fig. 3G is a blunt-end treated IR gene (4.6 kbp) of the DHFR locus Ori-3 region obtained above. Were prepared by inserting the EcoR I digest of ⁇ ⁇ I ⁇ ⁇ into the same or opposite direction with respect to BSR transcription.
  • pEPI—I (“Schaarschmidt, D., Baltin, J., Stehle, I. ⁇ , Li pps, HJ, and nippers, R. (2004) EMBO J. 23 (1), 191-201 ”, and“ Jenke, A. C, Stehle, IM, Herrmann, F., Eisenberger, ⁇ ⁇ , Baiker, A., Bode, J., Fackelmayer, F. 0., and Lipps, HJ (2004) Proc. Natl. Acad. Sci. USA 101, 11322—11327 ”) was provided by Daniel Schaarchmidt (Department of Biology, Universitat of Konstanz).
  • the pTHV shown in FIG. 3J was prepared by inserting the MAR of AR1 into the Kpn I site of ⁇ ⁇ ⁇ with a blunt end ligation.
  • pTH3 shown in FIG. 31 is blunt-end ligated to the Not I site of pTHV. It was created by inserting HSV poly A sequences with Chillon.
  • [0090] also includes a 2 ⁇ 4kbp c-myc IR partial fragment (C0 to C16) or a 4 ⁇ 6kbp DHFR gene locus Ori— / 3 region IR partial fragment (D;! To D11)
  • a vector was prepared by inserting a gene amplified by PCR using pNeo. Myc-2.4 and pSFVdhfr into a cage in the Asc I site of pTHV.
  • the base sequence of the 5 'primer (CO-5') for CO amplification used in the above PCR is SEQ ID NO: 26, and the base sequence of the 3 'primer (CO-3') is SEQ ID NO: 27. Indicated.
  • the base sequence of the 5 ′ primer (C 1-5 ′) for C1 amplification is shown in SEQ ID NO: 28, and the base sequence of the 3 primer (C 1-3 ′) is shown in SEQ ID NO: 29.
  • the base sequence of the 5 ′ primer (C2-5 ′) for C2 amplification is shown in SEQ ID NO: 30, and the base sequence of the 3 ′ primer (C2-3 ′) is shown in SEQ ID NO: 31.
  • the base sequence of the 5 ′ primer (C3-5 ′) for C3 amplification is shown in SEQ ID NO: 32
  • the base sequence of the 3 ′ primer (C3-3 ′) is shown in SEQ ID NO: 33
  • the base sequence of 5 ′ primer (C4 5 ′) for C4 amplification is shown in SEQ ID NO: 34
  • the base sequence of 3, primer (C4 3 ′) is shown in SEQ ID NO: 35.
  • the base sequence of the 5 ′ primer (C5-5 ′) for C5 amplification is shown in SEQ ID NO: 36
  • the base sequence of the 3 ′ primer (C5-3 ′) is shown in SEQ ID NO: 37.
  • the base sequence of the 5 ′ primer (C6-5 ′) for C6 amplification is shown in SEQ ID NO: 38
  • the base sequence of the 3 ′ primer (C 6-3 ′) is shown in SEQ ID NO: 39
  • the base sequence of the 5 ′ primer (C7-5 ′) for C7 amplification is shown in SEQ ID NO: 40
  • the base sequence of the 3 ′ primer (C7-3 ′) is shown in SEQ ID NO: 41.
  • the base sequence of the 5 ′ primer (C8-5 ′) for C8 amplification is shown in SEQ ID NO: 42
  • the base sequence of the 3 ′ primer (C8-3 ′) is shown in SEQ ID NO: 43.
  • the base sequence of the 5 ′ primer (C9 5 ′) for C9 amplification is shown in SEQ ID NO: 44
  • the base sequence of the 3 ′ primer (C9 3 ′) is shown in SEQ ID NO: 45
  • the base sequence of the 5 ′ primer (C10-5 ′) for C10 amplification is shown in SEQ ID NO: 46
  • the base sequence of the 3 ′ primer (C10-3 ′) is shown in SEQ ID NO: 47.
  • the base sequence of the 5 ′ primer (C11-5 ′) for C11 amplification is shown in SEQ ID NO: 48
  • the base sequence of the 3 ′ primer (C11-3 ′) is shown in SEQ ID NO: 49.
  • nucleotide sequence of the 5 ′ primer (C 12-5 ′) for C12 amplification is shown in SEQ ID NO: 50
  • nucleotide sequence of the 3 ′ primer (C 12-3 ′) is shown in SEQ ID NO: 51
  • base sequence of the 5 ′ primer (C13-5 ′) for C13 amplification is shown in SEQ ID NO: 52
  • base sequence of the 3 ′ primer (C13-3 ′) is shown in SEQ ID NO: 53. did.
  • the base sequence of 5 ′ primer (C14-5 ′) for C14 amplification is shown in SEQ ID NO: 54
  • the base sequence of 3 and primer (C14-3 ′) is shown in SEQ ID NO: 55.
  • nucleotide sequence of the 5 ′ primer (C 15-5 ′) for C15 amplification is shown in SEQ ID NO: 56
  • nucleotide sequence of the 3 ′ primer (C 15-3 ′) is shown in SEQ ID NO: 57.
  • the base sequence of the 5 'primer (C16-5') for C16 amplification is shown in SEQ ID NO: 58
  • the base sequence of the 3 'primer (C16-3') is shown in SEQ ID NO: 59.
  • the base sequence of the 5 ′ primer (D1-5 ′) for D1 amplification is shown in SEQ ID NO: 60
  • the base sequence of the 3 ′ primer (D1-3 ′) is shown in SEQ ID NO: 61
  • the base sequence of the 5 ′ primer (D2-5 ′) for D2 amplification is shown in SEQ ID NO: 62
  • the base sequence of the primer 3 (D2-3 ′) is shown in SEQ ID NO: 63
  • the base sequence of the 5 ′ primer (D3-5 ′) for D3 amplification is shown in SEQ ID NO: 64
  • the base sequence of the 3 ′ primer (D3-3 ′) is shown in SEQ ID NO: 65.
  • the base sequence of the 5 ′ primer (D4-5 ′) for D4 amplification is shown in SEQ ID NO: 66, and the base sequence of the 3 ′ primer (D4-3 ′) is shown in SEQ ID NO: 67.
  • the base sequence of the 5 ′ primer (D5-5 ′) for D5 amplification is shown in SEQ ID NO: 68
  • the base sequence of the 3 ′ primer (D5-3 ′) is shown in SEQ ID NO: 69.
  • the base sequence of the 5 ′ primer (D6-5 ′) for D6 amplification is shown in SEQ ID NO: 70
  • the base sequence of the 3 ′ primer (D6-3 ′) is shown in SEQ ID NO: 71.
  • the base sequence of 5 ′ primer (D7-5 ′) for D7 amplification is shown in SEQ ID NO: 72
  • the base sequence of 3 ′ primer (D7-3 ′) is shown in SEQ ID NO: 73
  • the base sequence of the 5 ′ primer (D8-5 ′) for D8 amplification is shown in SEQ ID NO: 74
  • the base sequence of the 3 ′ primer (D8-3 ′) is shown in SEQ ID NO: 75.
  • the base sequence of the 5 ′ primer (D9-5 ′) for D9 amplification is shown in SEQ ID NO: 76
  • the base sequence of 3 ′ primer (D9-3 ′) is shown in SEQ ID NO: 77.
  • the base sequence of the 5 ′ primer (D10-5 ′) for D10 amplification is shown in SEQ ID NO: 78
  • the base sequence of the 3 ′ primer (D10-3 ′) is shown in SEQ ID NO: 79.
  • the base sequence of the 5 ′ primer (D11-5 ′) for Dl 1 amplification is shown in SEQ ID NO: 80
  • the base sequence of the 3 ′ primer (Dl 1-3 ′) is shown in SEQ ID NO: 81.
  • the base sequence of CO amplified by the PCR is shown in SEQ ID NO: 82.
  • the base sequence of C1 amplified by the above PCR is shown in SEQ ID NO: 83.
  • the base sequence of C2 amplified by the PCR is shown in SEQ ID NO: 84.
  • the base sequence of C3 amplified by the PCR is shown in SEQ ID NO: 85.
  • the base sequence of C4 amplified by the above PCR is shown in SEQ ID NO: 86.
  • the base sequence of C5 amplified by the PCR is shown in SEQ ID NO: 87.
  • the base sequence of C6 amplified by the PCR is shown in SEQ ID NO: 88.
  • the base sequence of C7 amplified by the above-mentioned PCR is shown in SEQ ID NO: 89.
  • the base sequence of C8 amplified by the above PCR is shown in SEQ ID NO: 90.
  • the base sequence of C9 amplified by the PCR is shown in SEQ ID NO: 91.
  • the base sequence of C10 amplified by the PCR is shown in SEQ ID NO: 92.
  • the base sequence of C11 amplified by the PCR is shown in SEQ ID NO: 93.
  • the base sequence of C12 amplified by the PCR is shown in SEQ ID NO: 94.
  • the base sequence of C13 amplified by the PCR is shown in SEQ ID NO: 95.
  • the base sequence of C14 amplified by the PCR is shown in SEQ ID NO: 96.
  • the base sequence of C15 amplified by the PCR is shown in SEQ ID NO: 97.
  • the base sequence of C16 amplified by the above PCR is shown in SEQ ID NO: 98.
  • the base sequence of D1 amplified by the PCR is shown in SEQ ID NO: 99.
  • the base sequence of D2 amplified by the PCR is shown in SEQ ID NO: 100.
  • the base sequence of D3 amplified by the PCR is shown in SEQ ID NO: 101.
  • the base sequence of D4 amplified by the PCR is shown in SEQ ID NO: 102.
  • the base sequence of D5 amplified by the PCR is shown in SEQ ID NO: 103.
  • the base sequence of D6 amplified by the PCR is shown in SEQ ID NO: 104.
  • SEQ ID NO: 105 shows the base sequence IJ of D7 amplified by the PCR.
  • the base sequence of D8 amplified by the PCR is shown in SEQ ID NO: 106.
  • nucleotide sequence of D9 amplified by the PCR is shown in SEQ ID NO: 107.
  • the base sequence of D10 amplified by the PCR is shown in SEQ ID NO: 108.
  • the base sequence of D11 amplified by the PCR is shown in SEQ ID NO: 109.
  • the gene introduction method used in this example and the like is as follows. First, a plasmid used for gene transfer was purified from E. coli using a Qiagen plasmid purification kit (Qiagen Inc., Valencia, Calif.). Furthermore, when introducing the above plasmid into a cell, endotoxin derived from Escherichia coli mixed in the process of DNA purification is protected with MiraCLEAN (registered trademark) en After removal using the dotoxin removal kit (Mirus., Madison, WI), the gene is transferred to the cells using the GenePorter® 2 lipofection kit (Gene Therapy Systems, San Diego, CA) according to the manufacturer's recommended procedure. Introduced.
  • a Qiagen plasmid purification kit Qiagen Inc., Valencia, Calif.
  • MiraCLEAN registered trademark
  • the gene is transferred to the cells using the GenePorter® 2 lipofection kit (Gene Therapy Systems, San Diego, CA) according to the manufacturer's recommended procedure.
  • the gene-introduced cell is a human colon cancer cell line, COLO 320DM or COLO320 HSR, or a human cervical cancer cell line, Hela.
  • the above cell line was obtained from the place described in “Shimizu, et al. (2001) Cancer Research, vol. 61, p6987_6990” and cultured under the same conditions as described above.
  • COLO 320DM has many endogenous DMs due to the amplification of the c myc gene, while COLO 320HSR, an isogenic line of COLO 320DM, has more HSRs than DM! /.
  • the transformed cells were selected by culturing the cells in a selective medium supplemented with blasticidin to a final concentration of 5 11 g / ml 2 days after gene transfer.
  • the selective medium half of the selective medium in culture was replaced with freshly prepared selective medium every 3 to 5 days.
  • the FISH method the preparation of a probe for detecting a transgene used in the FISH method, and metaphase spreading are performed in culture after 4, 6 or 8 weeks of culture, respectively. A part of the cells was collected and carried out according to the method described in “Shimizu, et al. (2001) Cancer Research, vol. 61, p6987_6990”.
  • the probe is biotinized and can be detected by streptavidin bound with FITC (fluorescein isothiocyanate) that emits green fluorescence.
  • FITC fluorescein isothiocyanate
  • DNA was counterstained with PI (propidium iodide) that emits red fluorescence.
  • Inverted fluorescence microscope (M kon Plan Fluor, NA1.30 oil) equipped with an appropriate filter set and 100x objective lens (M kon Plan Fluor, NA1.30 oil) is used to detect fluorescent dyes on slide glass that has been fluorescently labeled by FISH ECLIPSE TE2000_U, Nikon) Using Fuji FinePix SI Pro digital camera (Fuji Film Co. Tokyo) connected to the above microscope, photo of the transgene and DNA in the cell is digital image Taken as.
  • Each obtained image was synthesized using image analysis software Adobe (registered trademark) Photpshop (registered trademark) version 4.0J (Adobe Systems Inc).
  • Plasmid pSFVdhfr, ⁇ ⁇ ⁇ 2 ⁇ dhfr, pTH2.dhfr, or pEPI—I COLO The gene was introduced into 320DM. When pSFVdhfr was introduced, transformed cells were collected after 8 weeks of culture, and when other plasmids were introduced, transformed cells were collected after 6 weeks of culture. DM and HSR were detected by FISH method for each collected transformed cell.
  • FIG. 2 shows the results of detection of DM and HSR by the FISH method.
  • a and “B” are the results of transformed cells into which pSFVdhfr was introduced
  • C is the result of transformed cells into which ⁇ ⁇ ⁇ 2.dhfr was introduced
  • D is the result of pTH2.dhfr.
  • E indicates the result of the transformed cell into which pEPI-I was introduced.
  • the arrowhead indicates DM and the arrow indicates HSR.
  • pTH. IR. MAR containing various combinations of IR and MAR was prepared, introduced into COLO 320 DM, and transformed cells were obtained after culturing for 4, 6 or 8 weeks.
  • the HSR generated in the transformed cells was examined by the FISH method, and the frequency of HSR (Frequency of HSR) was determined. The results are shown in FIG.
  • the frequency of HSR occurrence was calculated as “(number of transformed cells in which HSR occurred ⁇ number of transformed cells) ⁇ 100”.
  • “(10)” and “(1)” in the IR column in FIG. 4 indicate the case where IR is the same as the transfer direction of BSR and the opposite case, respectively.
  • “Eichi DNA fragment” indicates that the 4361 bp gene fragment of the phage is incorporated in place of IR, and nonej indicates that it does not contain IR.
  • HSR occurs only in transformed cells into which a plasmid having IR and MAR has been introduced. Furthermore, when a 43 61 bp gene fragment of ⁇ phage is used instead of IR, or when IR is not used, almost no HSR is generated. Therefore, there is a base sequence having activity to generate HSR in IR. It was suggested that there was.
  • Plasmid ( ⁇ ⁇ ⁇ . AR1, ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ . Dhfr, ⁇ ⁇ ⁇ ⁇ 2 ⁇ dhfr, ⁇ 2.dhfr, ⁇ 2.dhfr. Inv, pEPI-1, or ⁇ 3) Genes were introduced into COLO 320 HSR and COLO320DM. After the gene transfer, transformed cells were selected by culturing in a selective medium for 4 weeks or 8 weeks. Next, the frequency of HSR occurrence of the selected transformed cells was examined. The results are shown in FIG. [0103] According to FIG. 5, HSR occurred when p ⁇ ⁇ . Dhfr produced so that a collision occurred between gene replication and non-coding transcription was introduced.
  • ⁇ ⁇ ⁇ pAX created by adding one poly A sequence to prevent collision between non-coding transcription and gene duplication.
  • HSR was not generated. .
  • pTH2 Dhfr prepared so that there was no collision between non-coding transcription and gene replication was introduced, HSR occurred at a low frequency. This suggests that there was a collision between gene replication and non-coding transcription. From the above results, it was found that by adding a poly A sequence at both ends of the IR, the collision that occurs between gene replication and non-coding transcription can be eliminated.
  • HSR occurs in the transformed cells. On the other hand, if the incorporated partial fragment has no gene amplification activity, HSR will not be produced in the transformed cells.
  • the above identification method is referred to as “plasmid stabilization analysis method” for convenience.
  • pCO to pC16 a plasmid containing the full length of c-myc IR as a positive control ("pCf.lj"), or a c-myc IR as a negative control! /
  • Plasmid Plasmid
  • FIG. 6A is a schematic diagram of the c-myc locus (Genbank HSMYCC; accession number X00364).
  • the IR of the c-myc locus corresponds to the Hind ⁇ -Xho I fragment (2349 bp) of the c-myc locus.
  • Figures 6B and C show the positional relationship between the full IR length of the c-myc locus and its partial fragments, C0 to C16, and the frequency of HSR occurrence in transformed cells into which each partial fragment was introduced! / RU The “mouth (white square)” in FIG.
  • HSR occurrence frequency was highest in the transformed cells into which the plasmid (pCl 2) having a partial fragment containing the topoisomerasell binding region and Duplex Unwinding Element (DUE) was introduced.
  • the HSR frequency was higher than that of the positive control.
  • HSR was not formed in transformed cells into which a partial fragment that did not contain either the topoisomerasell binding region or Duplex Unwinding Element (DUE) was introduced, or when the frequency of HSR occurrence was extremely low.
  • topoisomerasell binding region and duplex unwinding element are essential elements for gene amplification!
  • DUE duplex Unwinding element
  • ⁇ Duplex Unwinding Element
  • DHFR locus Ori Shorte as Example 1 except that IR of region 3 was used. That is, using PCR, a partial fragment (D ;! to D11) of the DHFR locus Ori-3 region was prepared, and each partial fragment (D;! To D11) was recombined with the Asc of pTHV (Fig. The plasmid was inserted into the I site and a plasmid (pD;! To pDll) containing D;! To D11 was prepared (see various plasmids).
  • a plasmid containing the full length of IR in the DHFR locus Ori— / 3 region (“pDf. 1”) is adopted, and as a negative control, the IR in the DHFR locus Ori— / 3 region is not included! /, A plasmid (“pTHV”) was employed.
  • FIG. Fig. 7 shows a schematic diagram of the DHFR locus Ori— / 3 region (Genbank CFORIDHFR; accession number X94372).
  • the BamHI-Hind III fragment (4.6 kbp) of the DHFR locus Ori— ⁇ region is the DHFR gene.
  • Fig. 7 also shows the positional relationship between the IR full length of the DHFR locus Ori- / 3 region and its partial fragments D0 to D11, and transformed cells into which each partial fragment has been introduced. The frequency of HSR occurrence is shown.
  • transformed cells into which plasmids (pD6 and pD7) having a partial fragment lacking the region 3142 of the DHFR locus Ori- ⁇ region (Genbank CFORIDHFR; accession umber X94372) were introduced. About the power that HSR did not occur. In addition, the frequency of HSR was significantly reduced for transformed cells into which plasmids (pD5 and pDIO) having a partial fragment lacking the 4885th region of the DHFR locus Ori-3 region (Genbank CFORIDHFR; accession number X94372) were introduced. .
  • the region force S HSR at positions 3142 to 4885 in the DHFR locus Ori-3 region is essential.
  • the above region includes the topoisomerasell binding region (B in Fig. 7 (white square)) and Duplex Unwinding Element (DUE, ⁇ (black diamond) in Fig. 7). Therefore, in combination with the results of Example 1, it can be said that the IR partial fragment containing the topoisomerasell binding region and Duplex Unwinding Element (DUE) has gene amplification activity.
  • curved DNA (bent DNA, in Fig.
  • the SRa promoter motor (indicated as “PSR a” in FIG. 8A) in pC12 (shown in FIG. 8A) used in Example 1 was replaced with the SV40 early region-derived promoter region (hereinafter simply referred to as “SV40 promoter”).
  • SV40 promoter the SV40 early region-derived promoter region
  • PC12.Psv40 shown in FIG. 8B
  • the SV40 promoter is shown as “Psv40”.
  • the production method of pC12.Ps v40 is as follows.
  • ESMX linker oligonucleotide containing a multiple cloning site
  • a synthetic oligonucleotide containing a multiple cloning site was inserted into the excised site. Restriction enzyme of this multicloning site The site is located downstream of the ampicillin resistance gene (Amp R ) (ie, the site with the SRa promoter), and the restriction enzyme site is Eco RI-Sa 1 I-Mlu I -Xho from the 5 'end to the 3' end. Arranged in the order of I.
  • the base sequences of ESMX linkers are shown in Table 1, SEQ ID NO: 110 and SEQ ID NO: 111.
  • the plasmid prepared as described above was digested with restriction enzymes Sal I and Mlu I. Thereafter, the SV40 promoter digested with Sal I and Mlu I was inserted into the above plasmid to construct pC 12.Psv40. The SV40 promoter was inserted into the plasmid in a direction that transcribes BSR.
  • the SV40 promoter was prepared as follows. pMACS4.1 (Mitenyi Biotech Co., Ltd.) was used as a saddle and amplified by PCR using SV40 promoter amplification primers (MACS4.1 4288L and MACS4.1 1454R). The amplified SV40 promoter was used after restriction enzyme digestion with Sal I and Mlu I. Table 1 shows the base sequence of MACS4.1 4288L (SEQ ID NO: 1 12) and 54.1 1454R (SEQ ID NO: 13), which are primers for SV40 promoter amplification. The nucleotide sequence of SV40 promoter is shown in SEQ ID NO: 114, and the nucleotide sequence of SRa promoter is shown in SEQ ID NO: 115.
  • Anti-Pyrococcus kodakaraensis KOD mouse hyperpridoma cells that produce antibodies against vermilion DNA polymerase (obtained from: Biotechnology Industrial Research Institute, deposit number: FER M BP-6057), RNeasy Plus Mini Kit (QIAGEN) , 74134) to extract total RNA, and then use Rever Tra Ace- ⁇ - (manufactured by Sakai, FSK-101) to create single-stranded cD NA was synthesized.
  • PCR amplification using primers that specifically amplify the heavy and light chains excluding the signal sequence of the anti-KOD polymerase antibody, adding a signal sequence derived from the immunoglodarin ⁇ chain to each amplification product, and then a plasmid with a CMV promoter PCMV—H and pCMV-L were constructed by ligation to the Xb a I-Not I site.
  • pCMV—H and pCMV-L were cotransfected into Chinese nomstar ovary cells with pC12.Psv40. 2 days after transfection, Blasticidin (Invivogen, ant-b ⁇ 1) was added at a concentration of 5 g / ml, and cultured for 2 weeks while changing the medium every 3-4 days. A stable transformant was obtained.
  • Blasticidin Invivogen, ant-b ⁇ 1
  • p A BN.ARl having the full length IR of the DHFR locus Ori-3 region was used in place of pC12.Psv40 V, and IR / MAR was not transferred! / , Also went.
  • FIG. 9 is a bar graph showing the amount of antibody produced in each experimental group, and ⁇ with No IR / MAR '' shows the IR / MAR plasmid transfected. ⁇ ⁇ ⁇ BN.AR1 '' shows the result when co-transformation of p ⁇ BN.AR1 and ⁇ pC12.Psv40 '' co-transforms pC12.Psv40 The result of the case is shown.
  • Table 2 shows the amount of antibody production, the number of heavy chain and light chain antibody gene copies in each experimental group.
  • FIGS. 10 and 11 and Tables 3 and 4 The results of cloning using the limiting dilution method are shown in FIGS. 10 and 11 and Tables 3 and 4.
  • FIG. 10 and Table 3 are a bar graph and a table showing the amount of antibody protein produced for each clone when pC12.Psv40 is cotransformed.
  • FIG. 11 and Table 4 are a bar graph and a table showing the amount of antibody protein produced for each clone when pABN.ARl is cotransformed.
  • the vector (IRZMAR plasmid) according to the present invention contains a partial fragment of IR having gene amplification activity instead of the full length of IR. Therefore, it is smaller than the IR / MAR plasmid used in the conventional advanced gene amplification system.
  • the present invention can be used in various industries that produce proteins, such as pharmaceuticals, chemicals, foods, cosmetics, and fibers.

Abstract

La présente invention porte sur un vecteur utilisé pour l'amplification d'un gène cible dans une cellule de mammifère comprenant un fragment ayant une activité d'amplification, ce fragment étant un fragment partiel d'un domaine d'initiation de la réplication et comportant un site d'activité d'amplification de gène et un domaine de liaison de matrice nucléaire de mammifère. Lorsque le domaine d'initiation de réplication de mammifère tel que décrit précédemment prend naissance dans un locus c-myc, par exemple, ledit fragment partiel présenté ci-avant contient au moins un élément de déroulement duplex et un domaine de liaison de la topoisomérase II. Le vecteur décrit précédemment permet d'améliorer l'efficacité de transfert génique et l'efficacité d'amplification génique comparativement aux systèmes d'amplification génique poussée existants. Cette invention concerne notamment un procédé au moyen duquel on peut améliorer l'efficacité du transfert génique et l'efficacité d'amplification génique lors de l'amplification d'un gène cible au moyen d'un système d'amplification génique poussée ayant été mis au point par les inventeurs et un vecteur destiné à être utilisé dans le mode de réalisation de ce procédé.
PCT/JP2007/066133 2006-08-24 2007-08-20 Procédé d'amplification poussée d'un gène cible dans une cellule de mammifère et vecteur correspondant WO2008023671A1 (fr)

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KR1020097004529A KR101093835B1 (ko) 2006-08-24 2007-08-20 포유동물세포 내에서 목적 유전자를 고도로 증폭시키기 위한 방법 및 벡터
JP2008530901A JP5124737B2 (ja) 2006-08-24 2007-08-20 哺乳動物細胞内で目的遺伝子を高度に増幅させるための方法およびベクター
US12/438,545 US8137963B2 (en) 2006-08-24 2007-08-20 Method for highly amplifying target gene in mammalian cell and vector therefor
EP07792746A EP2058391B1 (fr) 2006-08-24 2007-08-20 Procédé d'amplification poussée d'un gène cible dans une cellule de mammifère et vecteur correspondant
CA2661647A CA2661647C (fr) 2006-08-24 2007-08-20 Procede d'amplification poussee d'un gene cible dans une cellule de mammifere et vecteur correspondant
DE602007009549T DE602007009549D1 (de) 2006-08-24 2007-08-20 Verfahren zur hohen amplifikation eines zielgens in einer säugerzelle und vektor dafür

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EP2058391A4 (fr) 2009-09-30
EP2058391A1 (fr) 2009-05-13
CA2661647C (fr) 2013-02-05
JP5124737B2 (ja) 2013-01-23
KR20090039820A (ko) 2009-04-22
US20100317060A1 (en) 2010-12-16
CA2661647A1 (fr) 2008-02-28
EP2058391B1 (fr) 2010-09-29
US8137963B2 (en) 2012-03-20
JPWO2008023671A1 (ja) 2010-01-07
DE602007009549D1 (de) 2010-11-11

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